Method of producing an exogenous protein in the egg of a chicken

ABSTRACT

This invention provides for proteins which are expressed in the avian oviduct, packaged into eggs laid by the avian and removed from the eggs.

This application is a continuation of application Ser. No. 11/274,674,filed Nov. 15, 2005, which is a continuation of application Ser. No.10/696,671, filed Oct. 28, 2003, now U.S. Pat. No. 7,521,591, issuedApr. 21, 2009, the disclosures of which are incorporated in theirentirety herein by reference, which is a continuation of applicationSer. No. 09/173,864, filed Oct. 16, 1998, now U.S. Pat. No. 6,730,822,issued May 4, 2004, the disclosure of which is incorporated in itsentirety herein by reference, which claims the benefit of U.S.Provisional Application No. 60/062,172, filed Oct. 16, 1997.

BACKGROUND OF THE INVENTION

a) Field of the Invention

The present invention relates to vectors and methods for theintroduction of exogenous genetic material into avian cells and theexpression of the exogenous genetic material in the cells. The inventionalso relates to transgenic avian species, including chickens, and toavian eggs which contain exogenous protein.

b) Description of Related Art

Numerous natural and synthetic proteins are used in diagnostic andtherapeutic applications; many others are in development or in clinicaltrials. Current methods of protein production include isolation fromnatural sources and recombinant production in bacterial and mammaliancells. Because of the complexity and high cost of these methods ofprotein production, however, efforts arc underway to developalternatives. For example, methods for producing exogenous proteins inthe milk of pigs, sheep, goats, and cows have been reported. Theseapproaches suffer from several limitations, including long generationtimes between founder and production transgenic herds, extensivehusbandry and veterinary costs, and variable levels of expressionbecause of position effects at the site of the transgene insertion inthe genome. Proteins are also being produced using milling and maltingprocesses from barley and rye. However, plant post-translationalmodifications differ from vertebrate post-translational modifications,which often have a critical effect on the function of the exogenousproteins.

Like tissue culture and mammary gland bioreactors, the avian oviduct canalso potentially serve as a bioreactor. Successful methods of modifyingavian genetic material such that high levels of exogenous proteins aresecreted in and packaged into eggs would allow inexpensive production oflarge amounts of protein. Several advantages of such an approach wouldbe: a) short generation times (24 weeks) and rapid establishment oftransgenic flocks via artificial insemination; b) readily scaledproduction by increasing flock sizes to meet production needs; c)post-translational modification of expressed proteins; 4) automatedfeeding and egg collection; d) naturally sterile egg whites and e)reduced processing costs due to the high concentration of protein in theegg white.

The avian reproductive system, including that of the chicken, is welldescribed. The egg of the hen consists of several layers which aresecreted upon the yolk during its passage through the oviduct. Theproduction of an egg begins with formation of the large yolk in theovary of the hen. The unfertilized oocyte is then positioned on top ofthe yolk sac. Upon ovulation or release of the yolk from the ovary, theoocyte passes into the infundibulum of the oviduct where it isfertilized if sperm are present. It then moves into the magnum of theoviduct which is lined with tubular gland. cells. These cells secretethe egg white proteins, including ovalbumin, lysozyme, ovomucoid,conalbumin, and ovornucin, into the lumen of the magnum where they aredeposited onto the avian embryo and yolk.

The ovalbumin gene encodes a 45 kD protein that is specificallyexpressed in the tubular gland cells of the magnum of the oviduct(Beato, Cell 56:335-344 (1989)). Ovalbumin is the most abundant eggwhite protein, comprising over 50 percent of the total protein producedby the tubular gland cells, or about 4 grams of protein per large GradeA egg (Gilbert, “Egg albumen and its formation” in Physiology andBiochemistry of the Domestic Fowl, Bell and Freeman, eds., AcademicPress, London, N.Y. pp. 1291-1329). The ovalbumin gene and over 20 kb ofeach flanking region have been cloned and analyzed (Lai et al., Proc.Natl. Acad. Sci. USA 75:2205-2209 (1978); Gannon et al., Nature278:428-424 (1979); Roop et al., Cell 19:63-68 (1980); and Royal et al.,Nature 279:125-132 (1975)).

Much attention has been paid to the regulation of the ovalbumin gene.The gene responds to steroid hormones such as estrogen, glucocorticoids,and progesterone, which induce the accumulation of about 70,000ovalbumin mRNA transcripts per tubular gland cell in immature chicks and100,000 ovalbumin mRNA transcripts per tubular gland cell in the maturelaying hen (Palmiter, J Biol. Chem. 248:8260-8270 (1973); Palmiter, Cell4:189-197 (1975)). DNAse hypersensitivity analysis and promoter-reportergene assays in transfected tubular gland cells defined a 7.4 kb regionas containing sequences required for ovalbumin gene expression. This 5′flanking region contains four DNAse I-hypersensitive sites centered at−0.25, −0.8, −3.2, and −6.0 kb from the transcription start site. Thesesites are called HS-I, -II, -III, and -IV, respectively. These regionsreflect alterations in the chromatin structure and are specificallycorrelated with ovalbumin gene expression in oviduct cells (Kaye et al.,EMBO 3:1137-1144 (1984)). Hypersensitivity of HS-II and -III areestrogen-induced, supporting a role for these regions inhormone-induction of ovalbumin gene expression.

HS-I and HS-II are both required for steroid induction of ovalbumin genetranscription, and a 1.4 kb portion of the 5′ region that includes theseelements is sufficient to drive steroid-dependent ovalbumin expressionin explanted tubular gland cells (Sanders and McKnight, Biochemistry 27:6550-6557 (1988)). HS-I is termed the negative-response element (“NRE”)because it contains several negative regulatory elements which repressovalbumin expression in the absence of hormone (Haekers et al., Mol.Endo. 9:1113-1126 (1995)). Protein factors bind these elements,including some factors only found in oviduct nuclei suggesting a role intissue-specific expression. HS-II is termed the steroid-dependentresponse element (“SDRE”) because it is required to promote steroidinduction of transcription. It binds a protein or protein complex knownas Chirp-I. Chirp-I is induced by estrogen and turns over rapidly in thepresence of cyclohexamide (Dean et al., Mol. Cell. Biol. 16:2015-2024(1996)). Experiments using an explanted tubular gland cell culturesystem defined an additional set of factors that bind SDRE in asteroid-dependent manner, including a NFκB-like factor (Nordstrom etal., J Biol. Chem. 268:13193-13202 (1993); Schweers and Sanders, J.Biol. Chem. 266: 10490-10497 (1991)).

Less is known about the function of HS-III and -IV. HS-III contains afunctional estrogen response element, and confers estrogen inducibilityto either the ovalbumin proximal promoter or a heterologous promoterwhen co-transfected into HeLa cells with an estrogen receptor cDNA.These data imply that HS-III may play a functional role in the overallregulation of the ovalbumin gene. Little is known about the function ofHS-IV, except that it does not contain a functional estrogen-responseelement (Kato et al., Cell 68: 731-742 (1992)).

There has been much interest in modifying eukaryotic genomes byintroducing foreign genetic material and/or by disrupting specificgenes. Certain eukaryotic cells may prove to be superior hosts for theproduction of exogenous eukaryotic proteins. The introduction of genesencoding certain proteins also allows for the creation of new phenotypeswhich could have increased economic value. In addition, somegenetically-caused disease states may be cured by the introduction of aforeign gene that allows the genetically defective cells to express theprotein that it can otherwise not produce. Finally, modification ofanimal genomes by insertion or removal of genetic material permits basicstudies of gene function, and ultimately may permit the introduction ofgenes that could be used to cure disease states, or result in improvedanimal phenotypes.

Transgenesis has been accomplished in mammals by several differentmethods. First, in mammals including the mouse, pig, goat, sheep andcow, a transgene is microinjected into the pronucleus of a fertilizedegg, which is then placed in the uterus of a foster mother where itgives rise to a founder animal carrying the transgene in its germline.The transgene is engineered to carry a promoter with specific regulatorysequences directing the expression of the foreign protein to aparticular cell type. Since the transgene inserts randomly into thegenome, position effects at the site of the transgene's insertion intothe genome may variably cause decreased levels of transgene expression.This approach also requires characterization of the promoter such thatsequences necessary to direct expression of the transgene in the desiredcell type are defined and included in the transgene vector (Hogan et al.Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory, NY(1988)).

A second method for effecting animal transgenesis is targeted genedisruption, in which a targeting vector bearing sequences of the targetgene flanking a selectable marker gene is introduced into embryonic stem(“ES”) cells. Via homologous recombination, the targeting vectorreplaces the target gene sequences at the chromosomal locus or insertsinto interior sequences preventing expression of the target geneproduct. Clones of ES cells bearing the appropriately disrupted gene areselected and then injected into early stage blastocysts generatingchimeric founder animals, some of which bear the transgene in the germline. In the case where the transgene deletes the target locus, itreplaces the target locus with foreign DNA borne in the transgenevector, which consists of DNA encoding a selectable marker useful fordetecting transfected ES cells in culture and may additionally containDNA sequences encoding a foreign protein which is then inserted in placeof the deleted gene such that the target gene promoter drives expressionof the foreign gene (U.S. Pat. Nos. 5,464,764 and 5,487,992 (M.P.Capecchi and K.R. Thomas)). This approach suffers from the limitationthat ES cells are unavailable in many mammals, including goats, cows,sheep and pigs. Furthermore, this method is not useful when the deletedgene is required for survival or proper development of the organism orcell type.

Recent developments in avian transgenesis have allowed the modificationof avian genomes. Germ-line transgenic chickens may be produced byinjecting replication-defective retrovirus into the subgerminal cavityof chick blastoderms in freshly laid eggs (U.S. Pat. No. 5,162,215;Bosselman et al., Science 243:533-534 (1989); Thoraval et al.,Transgenic Research 4:369-36 (1995)). The retroviral nucleic acidcarrying a foreign gene randomly inserts into a chromosome of theembryonic cells, generating transgenic animals, some of which bear thetransgene in their germ line. Unfortunately, retroviral vectors cannotharbor large pieces of DNA, limiting the size and number of foreigngenes and foreign regulatory sequences that may be introduced using thismethod. In addition, this method does not allow targeted introduction ordisruption of a gene by homologous recombination. Use of insulatorelements inserted at the 5′ or 3′ region of the fused gene construct toovercome position effects at the site of insertion has been described(Chim et al., Cell 74:504-514 (1993)).

In another approach, a transgene has been microinjected into thegerminal disc of a fertilized egg to produce a stable transgenic founderbird that passes the gene to the F1 generation (Love et al.Bio/Technology 12:60-63 (1994)). This method has several disadvantages,however. Hens must be sacrificed in order to collect the fertilized egg,the fraction of transgenic founders is low, and injected eggs requirelabor intensive in vitro culture in surrogate shells.

In another approach, blastodermal cells containing presumptiveprimordial germ cells (“PGCs”) are excised from donor eggs, transfectedwith a transgene and introduced into the subgerminal cavity of recipientembryos. The transfected donor cells are incorporated into the recipientembryos generating transgenic embryos, some of which are expected tobear the transgene in the germ line. The transgene inserts in randomchromosomal sites by nonhomologous recombination. This approach requirescharacterization of the promoter such that sequences necessary to directexpression of the transgene in the desired cell type are defined andincluded in the transgene vector. However, no transgenic founder birdshave yet been generated by this method.

Wentworth, Poult. Sci. 68:999-1010 (1989), used a targeting vectorcontaining flanking DNA sequences of the vitellogenin gene to deletepart of the resident gene in chicken blastodermal cells in culture.However, it has not been demonstrated that these cells can contribute tothe germ line and thus produce a transgenic embryo. In addition, thismethod is not useful when the deleted gene is required for survival orproper development of the organism or cell type.

Thus, it can be seen that there is a need for a method of introducingforeign DNA which is operably linked to a magnum-active promoter intothe avian genome. There is also a need for a method of introducingforeign DNA into nonessential portions of a target gene of the aviangenome such that the target gene's regulatory sequences drive expressionof the foreign DNA, preferably without disrupting the function of thetarget gene. The ability to effect expression of the integratedtransgene selectively within the avian oviduct is also desirable.Furthermore, there exists a need to create germ-line modified transgenicbirds which express exogenous genes in their oviducts and secrete theexpressed exogenous proteins into their eggs.

SUMMARY OF THE INVENTION

This invention provides methods for the stable introduction of exogenouscoding sequences into the genome of a bird and expressing thoseexogenous coding sequences to produce desired proteins or to alter thephenotype of the bird. Synthetic vectors useful in the methods are alsoprovided by the present invention, as are transgenic birds which expressexogenous protein and avian eggs containing exogenous protein.

In one embodiment, the present invention provides methods for producingexogenous proteins in specific tissues of avians. In particular, theinvention provides methods of producing exogenous proteins in an avianoviduct. Transgenes are introduced into embryonic blastodermal cells,preferably near stage X, to produce a transgenic bird, such that theprotein of interest is expressed in the tubular gland cells of themagnum of the oviduct, secreted into the lumen, and deposited onto theegg yolk. A transgenic bird so produced carries the transgene in itsgerm line. The exogenous genes can therefore be transmitted to birds byboth artificial introduction of the exogenous gene into bird embryoniccells, and by the transmission of the exogenous gene to the bird'soffspring stably in a Mendelian fashion.

The present invention provides for a method of producing an exogenousprotein in an avian oviduct. The method comprises as a first stepproviding a vector that contains a coding sequence and a promoteroperably linked to the coding sequence, so that the promoter can effectexpression of the nucleic acid in the tubular gland cells of the magnumof an avian oviduct. Next, the vector is introduced into avian embryonicblastodermal cells, either freshly isolated, in culture, or in anembryo, so that the vector sequence is randomly inserted into the aviangenome. Finally, a mature transgenic avian which expresses the exogenousprotein in its oviduct is derived from the transgenic blastodermalcells. This method can also be used to produce an avian egg whichcontains exogenous protein when the exogenous protein that is expressedin the tubular gland cells is also secreted into the oviduct lumen anddeposited onto the yolk of an egg.

In one embodiment, the production of a transgenic bird by randomchromosomal insertion of a vector into its avian genome may optionallyinvolve DNA transfection of embryonic blastodermal cells which are theninjected into the subgerminal cavity beneath a recipient blastoderm. Thevector used in such a method has a promoter which is fused to anexogenous coding sequence and directs expression of the coding sequencein the tubular gland cells of the oviduct.

In an alternative embodiment, random chromosomal insertion and theproduction of a transgenic bird is accomplished by transduction ofembryonic blastodermal cells with replication-defective orreplication-competent retroviral particles carrying transgene RNAbetween the 5′ and 3′ LTRs of the retroviral vector. For instance, inone specific embodiment, an avian leukosis virus (ALV) retroviral vectoris used which comprises a modified pNLB plasmid containing an exogenousgene that is inserted downstream of a segment of the ovalbumin promoterregion. An RNA copy of the modified retroviral vector, packaged intoviral particles, is used to infect embryonic blastoderms which developinto transgenic birds. Alternatively, helper cells which produce theretroviral transducing particles are delivered to the embryonicblastoderm.

In one embodiment, the vector used in the methods of the inventioncontains a promoter which is magnum-specific. In this embodiment,expression of the exogenous coding sequence occurs only in the oviduct.Optionally, the promoter used in this embodiment may be a segment of theovalbumin promoter region. One aspect of the invention involvestruncating the ovalbumin promoter and/or condensing the criticalregulatory elements of the ovalbumin promoter so that it retainssequences required for high levels of expression in the tubular glandcells of the magnum of the oviduct, while being small enough that it canbe readily incorporated into vectors. For instance, a segment of theovalbumin promoter region may be used. This segment comprises the5′-flanking region of the ovalbumin gene. The total length of theovalbumin promoter segment may be from about 0.88 kb to about 7.4 kb inlength, and is preferably from about 0.88 kb to about 1.4 kb in length.The segment preferably includes both the steroid-dependent regulatoryelement and the negative regulatory element of the ovalbumin gene. Thesegment optionally also includes residues from the 5′untranslated region(5′UTR) of the ovalbumin gene. In an alternative embodiment, themagnum-specific promoter may be a segment of the promoter region of theconalbumin, ovomucoid, or ovomucin genes.

In another embodiment of the invention, the vectors integrated into theavian genome contain constitutive promoters which are operably linked tothe exogenous coding sequence. Alternatively, the promoter used in theexpression vector may be derived from that of the lysozyme gene, a geneexpressed in both the oviduct and macrophages.

If a constitutive promoter is operably linked to an exogenous codingsequence which is to be expressed in the oviduct, then the methods ofthe invention may also optionally involve providing a second vectorwhich contains a second coding sequence and a magnum-specific promoteroperably linked to the second coding sequence. This second vector isalso expressed in the tubular gland cells of the mature transgenicavian. In this embodiment, expression of the first coding sequence inthe magnum is directly or indirectly dependent upon the cellularpresence of the protein expressed by the second vector. Such a methodmay optionally include the use of a Cre-loxP system.

In an alternative embodiment, the production of the transgenic bird isaccomplished by homologous recombination of the transgene into aspecific chromosomal locus. An exogenous promoter-less minigene isinserted into the target locus, or endogenous gene, whose regulatorysequences then govern the expression of the exogenous coding sequence.This technique, promoter-less minigene insertion (PMGI), is not limitedto use with target genes directing oviduct-specific expression, and maytherefore be used for expression in any organ when inserted into theappropriate locus. In addition to enabling the production of exogenousproteins in eggs, the promoter-less minigene insertion method isamenable to applications in the poultry production and egg-layingindustries where gene insertions may enhance critical aviancharacteristics such as muscling, disease resistance, and livability orto reduce egg cholesterol.

One aspect of the present invention provides for a targeting vectorwhich may be used for promoter-less minigene insertion into a targetendogenous gene in an avian. This vector includes a coding sequence, atleast one marker gene, and targeting nucleic acid sequences. The markergene is operably linked to a constitutive promoter, such as the Xenopuslaevis ef-1α promoter, the HSV tk promoter, the CMV promoter, and theβ-actin promoter, and can be used for identifying cells which haveintegrated the targeting vector. The targeting nucleic acid sequencescorrespond to the sequences which flank the point of insertion in thetarget gene, and then direct insertion of the targeting vector into thetarget gene.

The present invention provides for a method of producing an exogenousprotein in specific cells in an avian. The method involves providing atargeting vector containing the promoter-less minigene. The targetingvector is designed to target an endogenous gene that is expressed in thespecific cells into avian embryonic blastodernal cells. The transgenicembryonic blastodermal cells are then injected into the subgerminalcavity beneath a recipient blastoderm or otherwise introduced into avianembryonic blastodermal cells. The targeting vector is integrated intothe target endogenous gene. The resulting bird then expresses theexogenous coding sequence under the control of the regulatory elementsof the target gene in the desired avian cells. This method may also beused for producing an avian egg that contains exogenous protein if amature transgenic bird is ultimately derived from the transgenicembryonic blastodermal cells. In the transgenic bird, the codingsequence is expressed in the magnum under the control of the regulatorysequences of a target gene, and the exogenous protein is secreted intothe oviduct lumen, so that the exogenous protein is deposited onto theyolk of an egg laid by the bird.

In one embodiment of the invention, the targeted endogenous gene is agene expressed in the tubular gland cells of the avian oviduct. Apreferred target endogenous gene for selective expression in the tubulargland cells is the ovalbumin gene (OV gene). While the invention isprimarily exemplified via use of the ovalbumin gene as a targetendogenous gene, other suitable endogenous genes may be used. Forexample, conalbumin, ovomucoid, ovomucin, and lysozyme may all be usedas target genes for the expression of exogenous proteins in tubulargland cells of an avian oviduct in accordance with the invention.

The point of insertion in a method involving promoter-less minigeneinsertion may be in the 5′ untranslated region of the target gene.Alternatively, if the targeting vector used for the insertion containsan internal ribosome entry element directly upstream of the codingsequence, then the point of insertion may be in the 3′ untranslatedregion of the target gene.

Another aspect of the invention provides for an avian egg which containsprotein exogenous to the avian species. Use of the invention allows forexpression of exogenous proteins in oviduct cells with secretion of theproteins into the lumen of the oviduct magnum and deposition upon theyolk of the avian egg. Proteins thus packaged into eggs may be presentin quantities of up to one gram or more per egg.

Other embodiments of the invention provide for transgenic birds, such aschickens or turkeys, which carry a transgene in the genetic material oftheir germ-line tissue. In one embodiment, the transgene comprises anexogenous gene operably linked to a promoter which optionally may bemagnum-specific. In this transgenic bird the exogenous gene is expressedin the tubular gland cells of the oviduct. In an alternative embodiment,the transgene instead comprises an exogenous gene which is positioned ineither the 5′ untranslated region or the 3′ untranslated region of anendogenous gene in a manner that allows the regulatory sequences of theendogenous gene to direct expression of the exogenous gene. In thisembodiment, the endogenous gene may optionally be ovalbumin, lysozyme,conalbumin, ovomucoid, or ovomucin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1( a) and 1(b) illustrate ovalbumin promoter expression vectorscomprising ovalbumin promoter segments and a coding sequence, gene Xwhich encodes an exogenous protein X.

FIGS. 2( a), 2(b), 2(c) and 2(d) illustrate retroviral vectors of theinvention comprising an ovalbumin promoter and a coding sequence, geneX, encoding an exogenous protein X.

FIG. 2( e) illustrates a method of amplifying an exogenous gene forinsertion into the vectors of 2(a) and 2(b).

FIG. 2( f) illustrates a retroviral vector comprising an ovalbuminpromoter controlling expression of a coding sequence, gene X, and aninternal ribosome entry site (IRES) element enabling expression of asecond coding sequence, gene Y.

FIGS. 3( a) and 3(b) show schematic representations of the ALV-derivedvectors pNLB and pNLB-CMV-BL, respectively. The vectors are both shownas they would appear while integrated into the chicken genome.

FIG. 4 shows a graph showing the amount of β-lactamase found in the eggwhite of eggs from hens transduced with NLB-CMV-BL, as determined by theβ-lactamase activity assay.

FIG. 5 shows a western blot indicating the presence of β-lactamase inthe egg white of eggs from hens transduced with NLB-CMV-BL.

FIGS. 6( a) and 6(b) illustrate magnum-specific, recombination-activatedgene expression. Schematic cre and β-lactamase transgenes are shownintegrated into the genome of a hen in a non-magnum cell in FIG. 6( a).In FIG. 6( b), schematic cre recombinase and β-lactamase transgenes areshown integrated into the genome of a hen in a magnum cell.

FIG. 7 illustrates an alternative method of silencing β-lactamaseexpression using loxP sites in which two loxP sites flanking a stopcodon (TAA) in frame with the first codon (ATG) are inserted into theP-lactamase signal peptide coding sequence such that the signal peptideis not disrupted.

FIGS. 8( a) and 8(b) illustrate targeting vectors used for insertion ofa promoter-less minigene of the invention into a target gene.

FIG. 9 illustrates a targeting vector used for detecting correcthomologous insertion of a promoter-less minigene of the invention into atarget gene.

DETAILED DESCRIPTION OF THE INVENTION

a) Definitions and General Parameters

The following definitions are set forth to illustrate and define themeaning and scope of the various terms used to describe the inventionherein.

A “nucleic acid or polynucleotide sequence” includes, but is not limitedto, eukaryotic mRNA, cDNA, genomic DNA, and synthetic DNA and RNAsequences, comprising the natural nucleoside bases adenine, guanine,cytosine, thymidine, and uracil. The term also encompasses sequenceshaving one or more modified bases.

A “coding sequence” or “open reading frame” refers to a polynucleotideor nucleic acid sequence which can be transcribed and translated (in thecase of DNA) or translated (in the case of mRNA) into a polypeptide invitro or in vivo when placed under the control of appropriate regulatorysequences. The boundaries of the coding sequence are determined by atranslation start codon at the 5′ (amino) terminus and a translationstop codon at the 3′ (carboxy) terminus. A transcription terminationsequence will usually be located 3′ to the coding sequence. A codingsequence may be flanked on the 5′ and/or 3′ ends by untranslatedregions.

“Exon” refers to that part of a gene which, when transcribed into anuclear transcript, is “expressed” in the cytoplasmic mRNA after removalof the introns or intervening sequences by nuclear splicing.

Nucleic acid “control sequences” or “regulatory sequences” refer totranslational start and stop codons, promoter sequences, ribosomebinding sites, polyadenylation signals, transcription terminationsequences, upstream regulatory domains, enhancers, and the like, asnecessary and sufficient for the transcription and translation of agiven coding sequence in a defined host cell. Examples of controlsequences suitable for eukaryotic cells are promoters, polyadenylationsignals, and enhancers. All of these control sequences need not bepresent in a recombinant vector so long as those necessary andsufficient for the transcription and translation of the desired gene arepresent.

“Operably or operatively linked” refers to the configuration of thecoding and control sequences so as to perform the desired function.Thus, control sequences operably linked to a coding sequence are capableof effecting the expression of the coding sequence. A coding sequence isoperably linked to or under the control of transcriptional regulatoryregions in a cell when DNA polymerase will bind the promoter sequenceand transcribe the coding sequence into mRNA that can be translated intothe encoded protein. The control sequences need not be contiguous withthe coding sequence, so long as they function to direct the expressionthereof. Thus, for example, intervening untranslated yet transcribedsequences can be present between a promoter sequence and the codingsequence and the promoter sequence can still be considered “operablylinked” to the coding sequence.

The terms “heterologous” and “exogenous” as they relate to nucleic acidsequences such as coding sequences and control sequences, denotesequences that are not normally associated with a region of arecombinant construct or with a particular chromosomal locus, and/or arenot normally associated with a particular cell. Thus, a “heterologous”region of a nucleic acid construct is an identifiable segment of nucleicacid within or attached to another nucleic acid molecule that is notfound in association with the other molecule in nature. For example, aheterologous region of a construct could include a coding sequenceflanked by sequences not found in association with the coding sequencein nature. Another example of a heterologous coding sequence is aconstruct where the coding sequence itself is not found in nature (e.g.,synthetic sequences having codons different from the native gene).Similarly, a host cell transformed with a construct which is notnormally present in the host cell would be considered heterologous forpurposes of this invention.

“Exogenous protein” as used herein refers to a protein not naturallypresent in a particular tissue or cell, a protein that is the expressionproduct of an exogenous expression construct or transgene, or a proteinnot naturally present in a given quantity in a particular tissue orcell.

“Endogenous gene” refers to a naturally occurring gene or fragmentthereof normally associated with a particular cell.

The expression products described herein may consist of proteinaceousmaterial having a defined chemical structure. However, the precisestructure depends on a number of factors, particularly chemicalmodifications common to proteins. For example, since all proteinscontain ionizable amino and carboxyl groups, the protein may be obtainedin acidic or basic salt form, or in neutral form. The primary amino acidsequence may be derivatized using sugar molecules (glycosylation) or byother chemical derivatizations involving covalent or ionic attachmentwith, for example, lipids, phosphate, acetyl groups and the like, oftenoccurring through association with saccharides. These modifications mayoccur in vitro, or in vivo, the latter being performed by a host cellthrough posttranslational processing systems. Such modifications mayincrease or decrease the biological activity of the molecule, and suchchemically modified molecules are also intended to come within the scopeof the invention.

Alternative methods of cloning, amplification, expression, andpurification will be apparent to the skilled artisan. Representativemethods are disclosed in Sambrook, Fritsch, and Maniatis, MolecularCloning, a Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory(1989).

“PMGI” refers to promoter-less minigene insertion, a method in which agene lacking a promoter is inserted via homologous recombination into atarget gene such that the target gene's regulatory sequences govern theexpression of the inserted gene in an appropriate tissue. A minigene isa modified version of a gene, often just a cDNA with an appropriatepolyadenylation signal and sometimes an intron. A minigene usually lacksall of the introns of the genomic gene.

“Vector” means a polynucleotide comprised of single strand, doublestrand, circular, or supercoiled DNA or RNA. A typical vector may becomprised of the following elements operatively linked at appropriatedistances for allowing functional gene expression: replication origin,promoter, enhancer, 5′ mRNA leader sequence, ribosomal binding site,nucleic acid cassette, termination and polyadenylation sites, andselectable marker sequences. One or more of these elements may beomitted in specific applications. The nucleic acid cassette can includea restriction site for insertion of the nucleic acid sequence to beexpressed. In a functional vector the nucleic acid cassette contains thenucleic acid sequence to be expressed including translation initiationand termination sites. An intron optionally may be included in theconstruct, preferably >100 bp 5′ to the coding sequence.

In some embodiments the promoter will be modified by the addition ordeletion of sequences, or replaced with alternative sequences, includingnatural and synthetic sequences as well as sequences which may be acombination of synthetic and natural sequences. Many eukaryoticpromoters contain two types of recognition sequences: the TATA box andthe upstream promoter elements. The former, located upstream of thetranscription initiation site, is involved in directing RNA polymeraseto initiate transcription at the correct site, while the latter appearsto determine the rate of transcription and is upstream of the TATA box.Enhancer elements can also stimulate transcription from linkedpromoters, but many function exclusively in a particular cell type. Manyenhancer/promoter elements derived from viruses, e.g. the SV40, the Roussarcoma virus (RSV), and CMV promoters are active in a wide array ofcell types, and are termed “constitutive” or “ubiquitous.” The nucleicacid sequence inserted in the cloning site may have any open readingframe encoding a polypeptide of interest, with the proviso that wherethe coding sequence encodes a polypeptide of interest, it should lackcryptic splice sites which can block production of appropriate mRNAmolecules and/or produce aberrantly spliced or abnormal mRNA molecules.

The termination region which is employed primarily will be one ofconvenience, since termination regions appear to be relativelyinterchangeable. The termination region may be native to the intendednucleic acid sequence of interest, or may be derived from anothersource.

A vector is constructed so that the particular coding sequence islocated in the vector with the appropriate regulatory sequences, thepositioning and orientation of the coding sequence with respect to thecontrol sequences being such that the coding sequence is transcribedunder the “control” of the control or regulatory sequences. Modificationof the sequences encoding the particular protein of interest may bedesirable to achieve this end. For example, in some cases it may benecessary to modify the sequence so that it may be attached to thecontrol sequences with the appropriate orientation; or to maintain thereading frame. The control sequences and other regulatory sequences maybe ligated to the coding sequence prior to insertion into a vector.Alternatively, the coding sequence can be cloned directly into anexpression vector which already contains the control sequences and anappropriate restriction site which is in reading frame with and underregulatory control of the control sequences.

A “marker gene” is a gene which encodes a protein that allows foridentification and isolation of correctly transfected cells. Suitablemarker sequences include, but are not limited to green, yellow, and bluefluorescent protein genes (GFP, YFP, and BFP, respectively). Othersuitable markers include thymidine kinase (tk), dihydrofolate reductase(DHFR), and aminoglycoside phosphotransferase (APH) genes. The latterimparts resistance to the aminoglycoside antibiotics, such as kanamycin,neomycin, and geneticin. These, and other marker genes such as thoseencoding chloramphenicol acetyltransferase (CAT), β-lactamase,β-galactosidase (β-gal), may be incorporated into the primary nucleicacid cassette along with the gene expressing the desired protein, or theselection markers may be contained on separate vectors andcotransfected.

A “reporter gene” is a marker gene that “reports” its activity in a cellby the presence of the protein that it encodes.

A “retroviral particle”, “transducing particle”, or “transductionparticle” refers to a replication-defective or replication-competentvirus capable of transducing non-viral DNA or RNA into a cell.

The terms “transformation”, “transduction” and “transfection” all denotethe introduction of a polynucleotide into an avian blastodermal cell.

“Magnum” is that part of the oviduct between the infundibulum and theisthmus containing tubular gland cells that synthesize and secrete theegg white proteins of the egg.

A “magnum-specific” promoter, as used herein, is a promoter which isprimarily or exclusively active in the tubular gland cells of themagnum.

b) Transgenesis of Blastodermal Cells

By the methods of the present invention, transgenes can be introducedinto avian embryonic blastodermal cells, to produce a transgenicchicken, or other avian species, that carries the transgene in thegenetic material of its germ-line tissue. The blastodermal cells aretypically stage VII-XII cells, or the equivalent thereof, and preferablyare near stage X. The cells useful in the present invention includeembryonic germ (EG) cells, embryonic stem (ES) cells & primordial germcells (PGCs). The embryonic blastodermal cells may be isolated freshly,maintained in culture, or reside within an embryo.

A variety of vectors useful in carrying out the methods of the presentinvention are described herein. These vectors may be used for stableintroduction of an exogenous coding sequence into the genome of a bird.In alternative embodiments, the vectors may be used to produce exogenousproteins in specific tissues of an avian, and in the oviduct inparticular. In still further embodiments, the vectors are used inmethods to produce avian eggs which contain exogenous protein.

In some cases, introduction of a vector of the present invention intothe embryonic blastodermal cells is performed with embryonicblastodermal cells that are either freshly isolated or in culture. Thetransgenic cells are then typically injected into the subgerminal cavitybeneath a recipient blastoderm in an egg. In some cases, however, thevector is delivered directly to the cells of a blastodermal embryo.

In one embodiment of the invention, vectors used for transfectingblastodermal cells and generating random, stable integration into theavian genome contain a coding sequence and a magnum-specific promoter inoperational and positional relationship to express the coding sequencein the tubular gland cell of the magnum of the avian oviduct. Themagnum-specific promoter may optionally be a segment of the ovalbuminpromoter region which is sufficiently large to direct expression of thecoding sequence in the tubular gland cells. For instance, the promotermay be derived from the promoter regions of the ovalbumin, lysozyme,conalbumin, ovomucoid, or ovomucin genes. Alternatively, the promotermay be a promoter that is largely, but not entirely, specific to themagnum, such as the lysozyme promoter.

FIGS. 1( a) and 1(b) illustrate examples of ovalbumin promoterexpression vectors. Gene X is a coding sequence which encodes anexogenous protein. Bent arrows indicate the transcriptional start sites.In one example, the vector contains 1.4 kb of the 5′ flanking region ofthe ovalbumin gene (FIG. 1( a)). The sequence of the “−1.4kb promoter”of FIG. 1( a) corresponds to the sequence starting from approximately1.4kb upstream (−1.4kb) of the ovalbumin transcription start site andextending approximately 9 residues into the 5′ untranslated region ofthe ovalbumin gene. The approximately 1.4 kb-long segment harbors twocritical regulatory elements, the steroid-dependent regulatory element(SDRE) and the negative regulatory element (NRE). The NRE is so namedbecause it contains several negative regulatory elements which block thegene's expression in the absence of hormone. A shorter 0.88 kb segmentalso contains both elements. In another example, the vector containsapproximately 7.4 kb of the 5′ flanking region of the ovalbumin gene andharbors two additional elements (HS-III and HS-IV), one of which isknown to contain a functional region enabling induction of the gene byestrogen (FIG. 1 (b)). A shorter 6 kb segment also contains all fourelements and could optionally be used in the present invention.

Each vector used for random integration according to the presentinvention preferably comprises at least one 1.2 kb element from thechicken β-globin locus which insulates the gene within from bothactivation and inactivation at the site of insertion into the genome. Ina preferred embodiment, two insulator elements are added to one end ofthe ovalbumin gene construct. In the β-globin locus, the insulatorelements serve to prevent the distal locus control region (LCR) fromactivating genes upstream from the globin gene domain, and have beenshown to overcome position effects in transgenic flies, indicating thatthey can protect against both positive and negative effects at theinsertion site. The insulator element(s) are only needed at either the5′ or 3′ end of the gene because the transgenes are integrated inmultiple, tandem copies effectively creating a series of genes flankedby the insulator of the neighboring transgene. In another embodiment,the insulator element is not linked to the vector but is cotransfectedwith the vector. In this case, the vector and the element are joined intandem in the cell by the process of random integration into the genome.

Each vector may optionally also comprise a marker gene to allowidentification and enrichment of cell clones which have stablyintegrated the expression vector. The expression of the marker gene isdriven by a ubiquitous promoter that drives high levels of expression ina variety of cell types. In a preferred embodiment the green fluorescentprotein (GFP) reporter gene (Zolotukhin et al., J. Virol 70:4646-4654(1995)) is driven by the Xenopus elongation factor 1-α (ef-1-α) promoter(Johnson and Krieg, Gene 147:223-26 (1994)). The Xenopus ef-1α promoteris a strong promoter expressed in a variety of cell types. The GFPcontains mutations that enhance its fluorescence and is humanized, ormodified such that the codons match the codon usage profile of humangenes. Since avian codon usage is virtually the same as human codonusage, the humanized form of the gene is also highly expressed in avianblastodermal cells. In alternative embodiments, the marker gene isoperably linked to one of the ubiquitous promoters of HSV tk, CMV, or ,β-actin.

While human and avian codon usage is well matched, where a nonvertebrategene is used as the coding sequence in the transgene, the nonvertebrategene sequence may be modified to change the appropriate codons such thatcodon usage is similar to that of humans and avians.

Transfection of the blastodermal cells may be mediated by any number ofmethods known to those of ordinary skill in the art. The introduction ofthe vector to the cell may be aided by first mixing the nucleic acidwith polylysine or cationic lipids which help facilitate passage acrossthe cell membrane. However, introduction of the vector into a cell ispreferably achieved through the use of a delivery vehicle such as aliposome or a virus. Viruses which may be used to introduce the vectorsof the present invention into a blastodermal cell include, but are notlimited to, retroviruses, adenoviruses, adeno-associated viruses, herpessimplex viruses, and vaccinia viruses.

In one method of transfecting blastodermal cells, a packagedretroviral-based vector is used to deliver the vector into embryonicblastodermal cells so that the vector is integrated into the aviangenome.

As an alternative to delivering retroviral transduction particles to theembryonic blastodermal cells in an embryo, helper cells which producethe retrovirus can be delivered to the blastoderm.

A preferred retrovirus for randomly introducing a transgene into theavian genome is the replication-deficient ALV retrovirus. To produce anappropriate ALV retroviral vector, a pNLB vector is modified byinserting a region of the ovalbumin promoter and one or more exogenousgenes between the 5′ and 3′ long terminal repeats (LTRs) of theretrovirus genome. Any coding sequence placed downstream of theovalbumin promoter will be expressed at high levels and only in thetubular gland cells of the oviduct magnum because the ovalbumin promoterdrives the high level of expression of the ovalbumin protein and is onlyactive in the oviduct tubular gland cells. While a 7.4 kb ovalbuminpromoter has been found to produce the most active construct whenassayed in cultured oviduct tubular gland cells, the ovalbumin promotermust be shortened for use in the retroviral vector. In a preferredembodiment, the retroviral vector comprises a 1.4 kb segment of theovalbumin promoter; a 0.88 kb segment would also suffice.

Any of the vectors of the present invention may also optionally includea coding sequence encoding a signal peptide that will direct secretionof the protein expressed by the vector's coding sequence from thetubular gland cells of the oviduct. This aspect of the inventioneffectively broadens the spectrum of exogenous proteins that may bedeposited in avian eggs using the methods of the invention. Where anexogenous protein would not otherwise be secreted, the vector bearingthe coding sequence is modified to comprise a DNA sequence comprisingabout 60 bp encoding a signal peptide from the lysozyme gene. The DNAsequence encoding the signal peptide is inserted in the vector such thatit is located at the N-terminus of the protein encoded by the cDNA.

FIGS. 2( a)-2(d), and 2(f) illustrate examples of suitable retroviralvectors. The vector is inserted into the avian genome with 5′ and 3′flanking LTRs. Neo is the neomycin phosphotransferase gene. Bent arrowsindicate transcription start sites. FIGS. 2( a) and 2(b) illustrate LTRand oviduct transcripts with a sequence encoding the lysozyme signalpeptide (LSP), whereas FIGS. 2( c) and 2(d) illustrate transcriptswithout such a sequence. There are two parts to the retroviral vectorstrategy. Any protein that contains a eukaryotic signal peptide may becloned into the vectors depicted in FIGS. 2( b) and 2(d). Any proteinthat is not ordinarily secreted may be cloned into the vectorsillustrated in FIGS. 2( a) and 2(b) to enable its secretion from thetubular gland cells.

FIG. 2( e) illustrates the strategy for cloning an exogenous gene into alysozyme signal peptide vector. The polymerase chain reaction is used toamplify a copy of a coding sequence, gene X, using a pair ofoligonucleotide primers containing restriction enzyme sites that enablethe insertion of the amplified gene into the plasmid after digestionwith the two enzymes. The 5′ and 3′ oligonucleotides contain the Bsu36Iand Xba1restriction sites, respectively.

Another aspect of the invention involves the use of internal ribosomeentry site (IRES) elements in any of the vectors of the presentinvention to allow the translation of two or more proteins from a di- orpolycistronic mRNA. The IRES units are fused to 5′ ends of one or moreadditional coding sequences which are then inserted into the vectors atthe end of the original coding sequence, so that the coding sequencesare separated from one another by an IRES. Pursuant to this aspect ofthe invention, post-translational modification of the product isfacilitated because one coding sequence may encode an enzyme capable ofmodifying the other coding sequence product. For example, the firstcoding sequence may encode collagen which would be hydroxylated and madeactive by the enzyme encoded by the second coding sequence.

For instance, in the retroviral vector example of FIG. 2( f), aninternal ribosome entry site (IRES) element is positioned between twoexogenous coding sequences (gene X and gene y). The IRES allows bothprotein X and protein Y to be translated from the same transcriptdirected by the ovalbumin promoter. Bent arrows indicate transcriptionstart sites. The expression of the protein encoded by gene X is expectedto be highest in tubular gland cells, where it is specifically expressedbut not secreted. The protein encoded by gene Y is also expressedspecifically in tubular gland cells but because it is efficientlysecreted, protein Y is packaged into the eggs.

In another aspect of the invention, the coding sequences of vectors usedin any of the methods of the present invention are provided with a 3′untranslated region (3′ UTR) to confer stability to the RNA produced.When a 3′ UTR is added to a retroviral vector, the orientation of thefused ovalbumin promoter, gene X and the 3′ UTR must be reversed in theconstruct, so that the addition of the 3′ UTR will not interfere withtranscription of the full-length genomic RNA. In a presently preferredembodiment, the 3′ UTR may be that of the ovalbumin or lysozyme genes,or any 3′ UTR that is functional in a magnum cell, i.e. the SV40 lateregion.

In an alternative embodiment of the invention, a constitutive promoteris used to express the coding sequence of a transgene in the magnum of abird. In this case, expression is not limited to only the magnum;expression also occurs in other tissues within the avian. However, theuse of such a transgene is still suitable for effecting the expressionof a protein in the oviduct and the subsequent secretion of the proteininto the egg white if the protein is non-toxic to the avian in which itis expressed.

FIG. 3( a) shows a schematic of the replication-deficient avian leukosisvirus (ALV)-based vector pNLB, a vector which is suitable for use inthis embodiment of the invention. In the pNLB vector, most of the ALVgenome is replaced by the neomycin resistance gene (Neo) and the lacZgene, which encodes b-galactosidase. FIG. 3( b) shows the vectorpNLB-CMV-BL, in which lacZ has been replaced by the CMV promoter and theβ-lactamase coding sequence (β-La or BL). Construction of the vector isreported in the specific example, Example 1, below. β-lactamase isexpressed from the CMV promoter and utilizes a poly adenylation signal(pA) in the 3′ long terminal repeat (LTR). β-Lactamase has a naturalsignal peptide; thus, it is found in blood and in egg white.

Avian embryos have been successfully transduced with pNLB-CMV-BLtransduction particles (see specific examples, Example 2 and 3, below).The egg whites of eggs from the resulting stably transduced hens werefound to contain up to 20 mg of secreted, active β-lactamase per egg(see specific examples, Example 4 and 5, below).

In an alternative embodiment of the invention, transgenes containingconstitutive promoters are used, but the transgenes are engineered sothat expression of the transgene effectively becomes magnum-specific.Thus, a method for producing an exogenous protein in an avian oviductprovided by the present invention involves generating a transgenic avianthat bears two transgenes in its tubular gland cells. One transgenecomprises a first coding sequence operably linked to a constitutivepromoter. The second transgene comprises a second coding sequence thatis operably linked to a magnum-specific promoter, where expression ofthe first coding sequence is either directly or indirectly dependentupon the cellular presence of the protein expressed by the second codingsequence.

Optionally, site-specific recombination systems, such as the Cre-loxP orFLP-FRT systems, are utilized to implement the magnum-specificactivation of an engineered constitutive promoter. In one embodiment,the first transgene contains an FRT-bounded blocking sequence whichblocks expression of the first coding sequence in the absence of FTP,and the second coding sequence encodes FTP. In another embodiment, thefirst transgene contains a loxP-bounded blocking sequence which blocksexpression of the first coding sequence in the absence of the Creenzyme, and the second coding sequence encodes Cre. The loxP-boundedblocking sequence may be positioned in the 5′ untranslated region of thefirst coding sequence and the loxP-bounded sequence may optionallycontain an open reading frame.

For instance, in one embodiment of the invention, magnum-specificexpression is conferred on a constitutive transgene, by linking acytomegalovirus (CMV) promoter to the coding sequence of the protein tobe secreted (CDS) (FIGS. 6( a) and 6(b)). The 5′ untranslated region(UTR) of the coding sequence contains a loxP-bounded blocking sequence.The loxP-bounded blocking sequence contains two loxP sites, betweenwhich is a start codon (ATG) followed by a stop codon, creating a short,nonsense open reading frame (ORF). Note that the loxP sequence containstwo start codons in the same orientation. Therefore, to prevent themfrom interfering with translation of the coding sequence after loxPexcision, the loxP sites must be orientated such that the ATGs are inthe opposite strand.

In the absence of Cre enzyme, the cytomegalovirus promoter drivesexpression of the small open reading frame (ORF) (FIG. 6( a)). Ribosomeswill initiate at the first ATG, the start codon of the ORF, thenterminate without being able to reinitiate translation at the startcodon of the coding sequence. To be certain that the coding sequence isnot translated, the first ATG is out of frame with the coding sequence'sATG. If the Cre enzyme is expressed in cells containing the CMV-cDNAtransgene, the Cre enzyme will recombine the loxP sites, excising theintervening ORF (FIG. 6( b)). Now translation will begin at the startcodon of the coding sequence, resulting in synthesis of the desiredprotein.

To make this system tissue specific, the Cre enzyme is expressed underthe control of a tissue-specific promoter, such as the magnum-specificovalbumin promoter, in the same cell as the CMV-loxP-coding sequencetransgene (FIG. 6( b)). Although a truncated ovalbumin promoter may befairly weak, it is still tissue-specific and will express sufficientamounts of the Cre enzyme to induce efficient excision of theinterfering ORF. In fact, low levels of recombinase should allow higherexpression of the recombinant protein since it does not compete againstcoding sequence transcripts for translation machinery.

Alternate methods of blocking translation of the coding sequence includeinserting a transcription termination signal and/or a splicing signalbetween the loxP sites. These can be inserted along with the blockingORF or alone. In another embodiment of the invention, a stop codon canbe inserted between the loxP sites in the signal peptide of the codingsequence (see FIG. 7). Before recombinase is expressed, the peptideterminates before the coding sequence. After recombinase is expressed(under the direction of a tissue specific promoter), the stop codon isexcised, allowing translation of the coding sequence. The loxP site andcoding sequence are juxtaposed such that they are in frame and the loxPstop codons are out of frame. Since signal peptides are able to acceptadditional sequence (Brown et al., Mol. Gen. Genet. 197:351-7 (1984)),insertion of loxP or other recombinase target sequences (i.e. FRT) isunlikely to interfere with secretion of the desired coding sequence. Inthe expression vector shown in FIG. 7, the loxP site is present in thesignal peptide such that the amino acids encoded by loxP are not presentin the mature, secreted protein. Before Cre enzyme is expressed,translation terminates at the stop codon, preventing expression ofβ-lactamase. After recombinase is expressed (only in magnum cells), theloxP sites recombine and excise the first stop codon. Therefore,β-lactamase is expressed selectively only in magnum cells.

In the aforementioned embodiments, the blocking ORF can be any peptidethat is not harmful to chickens. The blocking ORF can also be a genethat is useful for production of the ALV-transduction particles and/ortransgenic birds. In one embodiment, the blocking ORF is a marker gene.

For instance, the blocking ORF could be the neomycin resistance gene,which is required for production of transduction particles. Once thetransgene is integrated into the chicken genome, the neomycin resistancegene is not required and can be excised.

Alternatively, β-lactamase can be used as the blocking ORF as it is anuseful marker for production of transgenic birds. (For specific examplesof the use of β-lactamase as a marker in transgenic birds, see Example4, below.) As an example, the blocking ORF in FIG. 6( a) is replaced byβ-lactamase and the downstream coding sequence now encodes a secretedbiopharmaceutical. β-Lactamase will be expressed in blood and othertissues; it will not be expressed in the magnum after magnum-specificexpression of Cre and recombination-mediated excision of β-lactamase,allowing expression of the desired protein.

The Cre and loxP transgenes could be inserted into the chicken genomevia mediated transgenesis either simultaneously or separately. Anymethod of transgenesis that results in stable integration into thechicken genome is suitable. Both the ovalbumin promoter-recombinase andCMV-loxP-CDS transgenes could be placed simultaneously into chickens.However, the efficiencies of transgenesis are low and therefore theefficiency of getting both transgenes into the chicken genomesimultaneously is low. In an alternative and preferred method, one flockis produced that carries the magnum-specific promoter/recombinasetransgene and a second is produced that carries the CMV-loxP-CDStransgene. The flocks would then be crossed to each other. Hensresulting from this outbreeding will express the coding sequence andonly in their magnum.

In an alternative method of transfecting blastodermal cells to produce atransgenic chicken, a targeting vector is used for promoter-lessminigene insertion (PMGI) into a target gene. The targeting vectorcomprises a coding sequence, at least one marker gene which is operablylinked to a constitutive promoter, and targeting nucleic acid sequenceswhich match the sequence flanking the desired point of insertion in thedesired target gene. The targeting nucleic acid sequences directinsertion of the targeting vector into the target gene. The length ofthese targeting sequences will vary. Each targeting sequence istypically at least about 1 kb in length, although longer sequences (upto 10 kb, for instance) may be preferred in some cases and shortersequences may be required in others. The marker gene allows for theidentification of cells which have integrated the targeting vector.

In one embodiment, the target gene is an endogenous gene that isexpressed in the avian oviduct. For instance, the target gene may beselected from the group consisting of ovalbumin, lysozyme, conalbumin,ovomucoid, and ovomucin. (It should be noted that because the lysozymegene is expressed in macrophages in addition to the oviduct cells, it isnot a suitable target gene when expression is desired to be restrictedonly to oviduct cells.)

PMGI may be used with target genes other than those expressed in theavian oviduct, and in species other than the avian species.

The point of insertion to which the vector is directed may be in eitherthe 5′ or 3′ untranslated region of the target gene. If the 3′untranslated region is targeted, then the targeting vector furthercomprises an internal ribosome entry site element positioned directlyupstream of the coding sequence on the vector.

FIG. 8( a) and 8(b) illustrate the insertion of PMGI into the 5′ or 3′untranslated region (UTR) of the ovalbumin target gene, respectively. Inthe embodiment illustrated in FIG. 8( a), a promoter-less minigene (PMG)is inserted into the 5′ UTR of the ovalbumin target gene. A dicistronicMRNA encoding both the exogenous protein and ovalbumin is transcribedfrom the transcription start site depicted by the arrow. Ribosomes bindto the 5′ end of the dicistronic mRNA, translate the exogenous gene,then terminate before translating the ovalbumin coding region. Note thatthe ovalbumin portion of the polycistronic transcript is not translated.Thus, the level of ovalbumin protein produced will be about half of thenormal level, as translation of one copy of the ovalbumin gene isdisrupted. In the embodiment illustrated in FIG. 8( b), the PMG isinserted into the 3′ UTR. Translation of the exogenous gene is initiatedby the presence of an IRES element to which the ribosome binds andtranslates the downstream coding region.

In either case, the targeting vectors contain a marker gene to enableidentification and enrichment of cell clones and populations which havestably integrated the targeting vectors. Suitable identification genesinclude but are not limited to neo, which encodes a protein conferringresistance to G418, or GFP, which encodes the green fluorescent protein(GFP). In a preferred embodiment, GFP expression is used to identifyclones uniformly fluorescing green and, therefore, containing a stablyintegrated targeting vector. The marker gene is expressed from aubiquitous promoter such as but not limited to the promoters of HSV tk,β-actin, CMV, or ef-1α. In a presently preferred embodiment, the ef-1αpromoter drives expression of GFP.

The present invention also provides for a vector which may be used forinsertion of a promoter-less minigene into a target gene, whichcomprises the elements of the targeting vector described above but alsoincludes a second marker gene which is operably linked to a secondconstitutive promoter. The second marker gene is positioned outside thetargeting nucleic acid sequences of the targeting vector, so that uponinsertion of the promoter-less minigene into the target gene, the secondmarker gene will not be inserted.

For instance, one embodiment of the invention involves use of markergenes encoding blue fluorescent protein (BFP) and GFP in the PMGItargeting vector (FIG. 9). This strategy is a variation of thepositive-negative selection strategy (U.S. Pat. Nos. 5,464,764 and5,487,992 (Capecchi et al.), in which BFP is used to identify the rarecells in which the promoter-less minigene (PMG) has correctly insertedinto the target gene. The BFP gene is inserted on the 3′ end of theoriginal targeting vector (See FIG. 9). When the targeting vector andtarget correctly undergo homologous recombination, only the GFP gene isinserted. Thus, colonies containing a correctly inserted PMG willfluoresce green. By contrast, in the majority of cells, the entirevector, including the BFP gene, will insert at random spots in thegenome. Colonies in which random insertion has taken place willfluoresce blue and green due to the presence of GFP and BFP.

Although FIG. 9 illustrates use of this vector in the 5′ UTR, thisvector is suitable for use in either the 5′ or 3′ UTR.

As mentioned above, the vectors produced according to the methods of theinvention may optionally be provided with a 3′ UTR containing apolyadenylation site to confer stability to the RNA produced. In apreferred embodiment, the 3′ UTR may be that of the exogenous gene, orselected from the group consisting of the ovalbumin, lysozyme, or SV40late region. However, the ovalbumin 3′ UTR is not suitable in a PMGIvector that is to be inserted into the endogenous ovalbumin gene becausethe addition of ovalbumin sequences to the PMGI vector will interferewith proper targeting.

c) Production of Exogenous Protein

Methods of the invention which provide for the production of exogenousprotein in the avian oviduct and the production of eggs which containexogenous protein involve an additional step subsequent to providing asuitable vector and introducing the vector into embryonic blastodermalcells so that the vector is integrated into the avian genome. Thesubsequent step involves deriving a mature transgenic avian from thetransgenic blastodermal cells produced in the previous steps. Deriving amature transgenic avian from the blastodermal cells optionally involvestransferring the transgenic blastodermal cells to an embryo and allowingthat embryo to develop fully, so that the cells become incorporated intothe bird as the embryo is allowed to develop. The resulting chick isthen grown to maturity. In an alterantive embodiment, the cells of ablastodermal embryo are transfected or transduced with the vectordirectly within the embryo. The resulting embryo is allowed to developand the chick allowed to mature.

In either case, the transgenic bird so produced from the transgenicblastodermal cells is known as a founder. Some founders will carry thetransgene in the tubular gland cells in the magnum of their oviducts.These birds will express the exogenous protein encoded by the transgenein their oviducts. If the exogenous protein contains the appropriatesignal sequences, it will be secreted into the lumen of the oviduct andonto the yolk of an egg.

Some founders are germ-line founders. A germ-line founder is a founderthat carries the transgene in genetic material of its germ-line tissue,and may also carry the transgene in oviduct magnum tubular gland cellsthat express the exogenous protein. Therefore, in accordance with theinvention, the transgenic bird will have tubular gland cells expressingthe exogenous protein and the offspring of the transgenic bird will alsohave oviduct magnum tubular gland cells that express the exogenousprotein. (Alternatively, the offspring express a phenotype determined byexpression of the exogenous gene in a specific tissue of the avian.)

The invention can be used to express, in large yields and at low cost, awide range of desired proteins including those used as human and animalpharmaceuticals, diagnostics, and livestock feed additives. Proteinssuch as human growth hormone, interferon, lysozyme, and β-casein areexamples of proteins which are desirably expressed in the oviduct anddeposited in eggs according to the invention. Other possible proteins tobe produced include, but are not limited to, albumin, α-1 antitrypsin,antithrombin III, collagen, factors VIII, IX, X (and the like),fibrinogen, hyaluronic acid, insulin, lactoferrin, protein C,erythropoietin (EPO), granulocyte colony-stimulating factor (G-CSF),granulocyte macrophage colony-stimulating factor (GM-CSF), tissue-typeplasminogen activator (tPA), feed additive enzymes, somatotropin, andchymotrypsin. Genetically engineered antibodies, such as immunotoxinswhich bind to surface antigens on human tumor cells and destroy them,can also be expressed for use as pharmaceuticals or diagnostics.

d) Examples

The following specific examples are intended to illustrate the inventionand should not be construed as limiting the scope of the claims.

Example 1 Vector Construction

The lacZ gene of pNLB, a replication-deficient avian leukosis virus(ALV)-based vector (Cosset et al., 1991), was replaced with anexpression cassette consisting of a cytomegalovirus (CMV) promoter andthe reporter gene, β-lactamase (β-La or BL). The pNLB and pNLB-CMV-BLvector constructs are diagrammed in FIGS. 3( a) and 3(b), respectively.

To efficiently replace the lacZ gene of pNLB with a transgene, anintermediate adaptor plasmid was first created, pNLB-Adapter.pNLB-Adapter was created by inserting the chewed back Apal/Apal fragmentof pNLB (Cosset et al., J Virol. 10 65:3388-94 (1991)) (in pNLB, the 5′ApaI resides 289 bp upstream of lacZ and the 3′ApaI resides 3′ of the 3′LTR and Gag segments) into the chewed-back KpnI/SacI sites ofpBluescriptKS(−). The filled-in MluI/XbaI fragment of pCMV-BL (Moore etal., Anal. Biochem. 247: 203-9 (1997)) was inserted into the chewed-backKpnI/NdeI sites of pNLB-Adapter, replacing lacZ with the CMV promoterand the BL gene (in pNLB, KpnI resides 67 bp upstream of lacZ and NdeIresides 100 bp upstream of the lacZ stop codon), thereby creatingpNLB-Adapter-CMV-BL. To create pNLB-CMV-BL, the HindIII/BlpI insert ofpNLB (containing lacZ) was replaced with the HindIII/BlpI insert ofpNLB-Adapter-CMV-BL. This two step cloning was necessary because directligation of blunt-ended fragments into the HindIII/BlpI sites of pNLByielded mostly rearranged subclones, for unknown reasons.

Example 2 Production of Transduction Particles

Sentas and Isoldes were cultured in F10 (Gibco), 5% newborn calf serum(Gibco), 1% chicken serum (Gibco), 50 μg/ml phleomycin (CaylaLaboratories) and 50 μg/ml hygromycin (Sigma). Transduction particleswere produced as described in Cosset et al., 1993, herein incorporatedby reference, with the following exceptions. Two days after transfectionof the retroviral vector pNLB-CMV-BL (from Example 1, above) into 9×10⁵Sentas, virus was harvested in fresh media for 6-16 hours and filtered.All of the media was used to transduce 3×10⁶ Isoldes in 3 100 mm plateswith polybrene added to a final concentration of 4 μg/ml. The followingday the media was replaced with media containing 50 μg/ml phleomycin, 50μg/ml hygromycin and 200 μg/ml G418 (Sigma). After 10-12 days, singleG418^(r) colonies were isolated and transferred to 24-well plates. After7-10 days, titers from each colony were determined by transduction ofSentas followed by G418 selection. Typically 2 out of 60 colonies gavetiters at 1-3×10⁵. Those colonies were expanded and virus concentratedto 2-7 ×10⁷ as described in Allioli et al., Dev. Biol. 165:30-7 (1994),herein incorporated by reference. The integrity of the CMV-BL expressioncassette was confirmed by assaying for β-lactamase in the media of cellstransduced with NLB-CMV-BL transduction particles.

Example 3 Production of Transgenic Chickens

Stage X embryos in freshly laid eggs were transduced with NLB-CMV-BLtransduction particles (from Example 2, above) as described in Thoravalet al., Transgenic Res. 4:369-377 (1995), herein incorporated byreference, except that the eggshell hole was covered with 1-2 layers ofeggshell membrane and, once dry, Duco model cement.

Approximately 120 White Leghorns were produced by transduction of thestage X embryos with NLB-CMV-BL transduction particles. These birdsconstitute chimeric founders, not fully transgenic birds. Extensiveanalysis of DNA in the blood and sperm from the transduced chickensindicates that 10-20% of the birds had detectable levels of thetransgene in any given tissue. Of those birds carrying the transgene,approximately 2-15% of the cells in any given tissue were actuallytransgenic.

Example 4 β-lactamase Activity Assay in Blood and Egg White

When hens produced in Example 3, above, began to lay eggs, the eggwhites of those eggs were assayed for the presence of β-lactamase. Theβ-lactamase assay was carried out as described in Moore et al., Anal.Biochem. 247:203-9 (1997), herein incorporated by reference, with thefollowing modifications.

To assay blood from two to ten day old chicks, the leg vein was prickedwith a scalpel. 50 μl of blood was collected in a heparinized capillarytube (Fisher), of which 25 μl was transferred to 100 μlphosphate-buffered saline (PBS) in a 96-well plate. Various dilutions ofpurified P-lactamase (Calbiochem) was added to some wells prior toaddition of blood from control (non-transduced) chicks to establish aβ-lactamase standard curve. After one day at 4° C., the plate wascentrifuged for 10 minutes at 730×g. 25 μl of the supernatant was addedto 75 μl of PBS. 100 μl of 20 μM7-(thienyl-2-acetamido)-3-[2-(4-N,N-dimethylaminophenylazo)pyridinium-methyl]-3-cephem-4-carboxylicacid (PADAC,from Calbiochem) in PBS was added, and the wells were readimmediately on a plate reader in a 10 minute kinetic read at 560 nm orleft overnight in the dark at room temperature. Wells were scoredpositive if the well had turned from purple to yellow. To assay bloodfrom older birds, the same procedure was followed except that 200-300 μlblood was drawn from the wing vein using a syringe primed with 50 μl ofheparin (Sigma).

Analysis of the NLB-CMV-BL transduced flock revealed nine chickens thathad significant levels of β-lactamase in their blood. Three of thesechickens were males and these were the only three males that hadsignificant levels of the NLB-CMV-BL transgene in their sperm asdetermined by PCR analysis (see Example 10, below). Thus, these are themales that are to be outbred to obtain fully transgenic G₁ offspring.The other six chickens were the hens that expressed β-lactamase in theirmagnum tissue (see below). Other birds had low levels of β-lactamase(just above the level of detection) in their blood but did not havetransgenic sperm or eggs containing β-lactamase. Thus β-lactamaseexpression in blood is a strong indicator of whether a chicken wassuccessfully transduced.

To assay β-lactamase in egg white, freshly laid eggs were transferredthat day to a 4° C. cooler, at which point the β-lactamase is stable forat least one month. (Bacterially-expressed, purified β-lactamase addedto egg white was determined to lose minimal activity over several weeksat 4° C., confirming the stability of β-lactamase in egg white.) Tocollect egg white samples, eggs were cracked onto plastic wrap. The eggwhite was pipetted up and down several times to mix the thick and thinegg whites. A sample of the egg white was transferred to a 96 wellplate. 10 μl of the egg white sample was transferred to a 96-well platecontaining 100 μl of PBS supplemented with 1.5 μl of 1 M NaH₂PO₄, pH 5.5per well. After addition of 100 μl of 20 μM PADAC, the wells were readimmediately on a plate reader in a 10 minute or 12 hour kinetic read at560 nm. Various dilutions of purified β-lactamase was added to somewells along 10 μl of egg white from control (non-transduced) hens toestablish a β-lactamase standard curve. Egg white from both untreatedand NLB-CMV-BL transduced hens were assayed for the presence ofβ-lactamase.

Significant levels of β-lactamase were detected in the egg white of sixhens, as shown in FIG. 4 and Table 1, below. Eggs laid by Hen 1522(“Betty Lu”), the first hen to demonstrate expression in eggs, have 0.3mg or higher of active β-lactamase per egg. Also shown is β-lactamaseproduction from three other NLB-CMV-BL transduced hens (Hen 1549, Hen1790 and Hen 1593). Every hen that laid eggs containing β-lactamase hadsignificant levels of β-lactamase in its blood.

TABLE 1 Expression of β-lactamase in eggs of NLB-CMV-BL treated hens.Hen # Average mg of β-lactamase per egg # of eggs assayed 1 Control  0.1± 0.07 29 2 1522 0.31 ± 0.07 20 3 1549 0.96 ± 0.15 22 4 1581 1.26 ± 0.1912 5 1587 1.13 ± 0.13 15 6 1790 0.68 ± 0.15 13 7 1793 1.26 ± 0.18 12

Control is eggs from untreated hens. The low level of BL in these eggsis due to spontaneous breakdown of PADAC during the course of thekinetic assay. The other hens were transduced with NLB-CMV-BL asdescribed in Example 3. Egg white from each egg was assayed intriplicate.

Based on the β-lactamase activity assay, the expression levels ofβ-lactamase appeared to range from 0.1 to 1.3 mg per egg (assuming 40milliliters of egg white per egg). However, these quantities weresignificantly lower from the quantities obtained by western blot assay(see Example 5, below) and were determined to be deceptively lower thanthe true values. The difference in results between the enzymaticactivity assay and the western blot analysis (Example 5) was found to bedue to the presence of a β-lactamase inhibitor in egg white. Theactivity of purified β-lactamase was shown to be inhibited by egg whitesuch that 50 ml of egg white in a 200 ml reaction resulted in nearly100% inhibition, whereas 10 ml of egg white in a 200 ml reactionresulted in only moderate inhibition. Furthermore, spontaneous breakdownof the enzymatic substrate, PADAC, during the course of the assay alsocontributed to the erroneously low calculation of β-lactamaseconcentration.

Example 5 Western Blot of β-Lactamase in Egg White

Western blot analysis of the same egg white as was assayed in Example 4confirmed the presence of β-lactamase and provided a more accuratemeasurement of the amount of β-lactamase present in the egg than thekinetic assay of Example 4, above.

To perform the analysis, 10 μl of egg white was added to 30 μl of 0.5 MTris-Cl, pH 6.8, 10% sodium dodecyl sulfate (SDS), 10% glycerol, 1.43 M2-mercaptoethanol, 0.001% bromophenol blue. Samples were heated to 95°C. for 5 min, separated on 12% SDS-PAGE and transferred to Immobilon Pmembranes (Millipore). β-lactamase was detected with 1:500 dilution ofrabbit anti-β-lactamase (5 Prime-3 Prime) and 1:5000 dilution of goatanti-rabbit IgG HRP conjugate (Promega). Immunoblots were visualizedwith the Enhanced Chemiluminescence (ECL) Western Blotting System(Amersham).

Various β-lactamase samples were analyzed by western blotting and anti-β-lactamase antibody. The results are shown in FIG. 5. Lanes 1-4 of theblot contain 5.2, 1.3, 0.325, and 0.08 μg, respectively, of bacteriallyexpressed, purified β-lactamase added to control egg white, forming astandard curve. Lane 5 contains control egg white from an untreated hen.In lane 6 is 2 μl of egg white from Hen 1522 (Betty Lu). Lanes 7-8contain 1 and 2 μls, respectively, of egg white from Hen 1790. Lanes9-10 contain 1 and 2 μls, respectively, of egg white from Hen 1793. 1and 2 μls aliquots of egg white from Hen 1549 was run in lanes 11-12.Lanes 13-14 show 1 and 2 μls, respectively, of egg white from Hen 1581.2 μls of egg white from Hen 1587 is shown in lane 15.

The position of molecular weight standards is noted in FIG. 5 to theleft of the blot in kilodaltons (kDa). The band at 31 kDa isβ-lactamase. The molecular weight of the β-lactamase in the egg white issimilar to that of purified β-lactamase. The egg white β-lactamase isalso a single molecular species, indicating that synthesis was faithfulto the β-lactamase coding sequence and that β-lactamase is very stablein magnum cells as well as egg white. The band at 13 kDa is an egg whiteprotein that cross-reacts with the anti- β-lactamase antibody.

Based on the western blot results, β-lactamase in lane 6 (from Hen 1522,Betty Lu) is estimated at 120 ng, or 2.4 mg per egg, assuming 40 mls ofegg white per egg. β-lactamase in lane 9 (from Hen 1793) is estimated at325 rig which corresponds to 13 mg per egg. The β-lactamase levels peregg as estimated by the western blot analysis were considerably higher(up to 10-fold higher) than the levels estimated by the β-lactamaseenzyme assay of Example 4. As explained above, the discrepancy in theprotein estimates is believed to be caused by inhibition of enzymeactivity by egg white and breakdown of the substrate.

It should be noted that the up to 13 mg of β-lactamase per egg reportedhere was produced by chimeric founders, not fully transgenic birds. Asreported above, only 2-15% of the cells in any given tissue of thechimeric founders were actually transgenic. Assuming that this extent ofmosaicism also applies to magnum tissue, then the magnums of the sixβ-lactamase egg-positive hens were only partially transgenic. Therefore,fully transgenic birds (G₁ offspring) would be expected to express muchhigher levels, possibly as high as 200 mg/egg. This estimate issignificant because it indicates that non-magnum specific promoters suchas CMV can effectively compete with magnum specific genes such asovalbumin and lysozyme for the egg-white protein synthesis machinery.

Example 6 Isolation and Ex Vivo Transfection of Blastodermal Cells

In an alternative embodiment of the invention, blastodermal cells aretransfected ex vivo with an expression vector.

In this method, donor blastodermal cells are isolated from fertilizedeggs of Barred Plymouth Rock hens using a sterile annular ring ofWhatman filter paper which is placed over a blastoderm and lifted aftercutting through the yolk membrane of the ring. The ring bearing theattached blastoderm is transferred to phosphate-buffered saline (PBS) ina petri dish ventral side up, and adhering yolk is removed by gentlepipetting. The area opaca is dissected away with a hair loop and thetranslucent stage X blastoderm is transferred via a large-bore pipettetip to a microfuge tube. About 30,000-40,000 cells are isolated perblastoderm and for a typical experiment 10 blastoderms are collected.

Cells are dispersed by brief trypsin (0.2%) digestion, washed once bylow speed centrifugation in Dulbecco's modified Eagle's medium (DMEM)and then transfected with linearized plasmids via lipofectin (16 mg/200ml, BRL) for 3 hours at room temperature. The vectors shown in FIG. 1,3, or 4 would serve as suitable expression constructs here. Cells arewashed free of lipofectin with medium and then 400-600 cells areinjected into g-irradiated (650 rads) recipient stage X embryos from theAthens-Canadian random-bred line (AC line). Injection is through a smallwindow (˜0.5 cm) into the subgerminal cavity beneath the recipientblastoderms. Windows are sealed with fresh egg shell membrane and Ducoplastic cement. Eggs are then incubated at 39.1° C. in a humidifiedincubator with 90° rotation every 2 hr.

Example 7 Identification of Transgenic Mosaics by PCR Assay

Among the chicks which hatch from embryos containing transfected ortransduced blastodermal cells, only those exhibiting Barred PlymouthRock feather mosaicism are retained. Even if no reporter gene is presentin the transgene, transgenic mosaics can be identified by PCR assay.

To identify transgenic mosaics, DNA blood and black feather pulp ofindividual chicks are assayed by PCR for the presence of the transgeneusing a primer pair specific to the transgene as described by Love etal., Bio/Technology 12:60-63 (1994). Transgene chimeras are induced,withdrawn and re-induced with diethylstilbestrol (DES) pellets andexcised magnums analyzed for expression of reporter activity. Blood andliver are assayed to monitor tissue specificity.

Male and female blood DNA was collected at 10 to 20 days post-hatch. TheDNA is extracted from the blood using a novel high-throughput method ofDNA extraction developed in our laboratory. In this method, blood isdrawn from a wing vein into a heparinized syringe and one drop isimmediately dispensed into one well of a flat-bottom 96-well dishcontaining a buffer which lyses cytoplasmic membranes exclusively. Theplate is then briefly centrifuged, which pellets the nuclei. Thesupernatant is removed and a second lysis buffer is added which releasesgenomic DNA from nuclei and degrades nucleases. The DNA is ethanolprecipitated in the plate, washed with 70% ethanol, dried andresuspended in 100 μl of water per well. As much as 80 μg of DNA can beobtained from one drop (8 μl) of chick blood. At least 768 samples canbe processed by one person in one day and the DNA is suitable for PCRand Taqman™ (Perkin Elmer/Applied Biosystems) analysis.

The isolated DNA is then tested for the presence of the transgenes usingthe Taqman™ sequence detection assay to evaluate the efficiency of theembryo transduction process. The Taqman™ sequence detection systemallows the direct detection of a specific sequence. Afluorescently-labeled oligonucleotide probe complementary to an internalregion of a desired PCR product only fluoresces when annealed to thedesired PCR product, which in this case is complementary to thetransgene. Because all of the detection occurs in the PCR tube duringthe cycling process, the Taqman™ system allows high-throughput PCR (nogel electrophoresis is needed) as well as sequence detection analogousto and as sensitive as Southern analysis. 1 μl of the isolated DNA,which contains 600-800 ng of DNA, is used for the Taqman™ reaction. Eachreaction contains two sets of primer pairs and Taqman™ probes. The firstset detects the chicken glyceraldehyde 3-phosphate dehydrogenase gene(GAPDH) and is used as an internal control for the quality of thegenomic DNA and also serves as a standard for quantitation of thetransgene dosage. The second set is specific for the desired transgene.Fluorescence is detected in a dissecting stereomicroscope equipped withepifluorescence detection. The two Taqman™ probes are attached todifferent dyes which fluoresce at unique wavelengths: thus both PCRproducts are detected simultaneously in an ABI/PE 7700 SequenceDetector. It is estimated that up to 180 birds will hatch, and 20% (36birds) will contain the transgene in their blood.

Example 8 Identification of Blastodermal Cells with a CorrectlyIntegrated Promoter-Less Minigene (PMG)

Following transfection with a PMGI targeting vector such as those shownin FIG. 8, cells are grown on a feeder line in conditioned medium toproduce colonies in which all or nearly half of the cells are uniformlygreen in fluorescence. Fluorescence is detected in a dissectingstereomicroscope equipped with epifluorescence detection. Uniformfluorescence indicates that the vector has stably integrated into thegenome. Of these cell clones, only a small subset actually have the PMGinserted correctly in the target gene. The majority of the clones havePMG integrated randomly into the genome. To identify clones containing acorrectly integrated PMG, colonies are screened using a Taqman™ PCRassay, as described above. Two primers are used to amplify a segment ofthe transgene at its site of integration. One primer lies in gene X, theexogenous gene to be expressed in the oviduct, and the other justoutside the 5′ targeting sequence, so that the fragment can only beamplified by correct insertion into the target gene. Colonies containinga correctly integrated transgene are subjected to limited passage inculture on feeder cells in the presence of a variety of cytokines thatpromote their growth in the absence of differentiation. Cells areinjected into recipient embryos. Alternatively, green colonies arepooled and injected into recipient embryos. Hatched chicks are screenedsubsequently for the presence of the correctly inserted transgene.

Example 9 Blue/Green Detection for Promoter-Less Minigene Insertion(PMGI)

Following transfection with a PMGI targeting vector like that of FIG. 4,cells are grown for one day in the absence of a feeder layer and greencells separated from blue/green cells using a fluorescence-activatedcell sorter the next day. Green cells are then briefly passaged onfeeder cells prior to injection into recipient embryos. Green cells arealso screened as above for correct insertion.

Example   Production of Fully Transgenic G₁ Chickens

Males are selected for breeding because a single male can give rise to20 to 30 G₁ offspring per week as opposed to 6 G₁ offspring per femaleper week, thereby speeding the expansion of G₁ transgenics. The feed ofG₀ males is supplemented with sulfamethazine, which accelerates thesexual maturation of males such that they can start producing sperm at10-12 weeks of age instead of 20-22 weeks without influencing theirhealth or fertility (Speksnijder and Ivarie, unpublished data).

Sperm DNA of all males are screened for the presence of the transgene.Sperm are collected and the DNA extracted using Chelex-100. Briefly, 3μl of sperm and 200 μl of 5% Chelex-100 are mixed, followed by additionof 2 μl of 10 mg/ml proteinase K and 7 μl of 2 M DTT. Samples areincubated at 56° C. for 30-60 minutes. Samples are boiled for 8 minutesand vortexed vigorously for 10 seconds. After centrifugation at 10 to 15kG for 2-3 minutes, the supernatant is ready for PCR or Taqman™analysis. The DNAs are analyzed by the Taqman™ assay using a Taqman™probe and primers complementary to the transgene. Of the 90 G₀ males, itis estimated that 5%, or 4 to 5, will have the transgene in their spermDNA.

As noted above in Example 4, the NLB-CMV-BL transduced flock includedthree males that had significant levels of the NLB-CMV-BL transgene intheir sperm as determined by PCR analysis (see Example 10). Thus, thesemales are chosen for further breeding to obtain fully transgenic G₁offspring.

By breeding germline transgenic males to 90 non-transgenic White Leghornfemales per week, it is estimated that 16 G₁ offspring per week will beobtained. Hatched chicks are vent-sexed and screened for the presence ofthe transgene in their blood DNA by the Taqman™ assay. Twenty male andfemale G₁ transgenics will be obtained or 40 total, which will take upto 3 weeks.

Males will be kept for further breeding and females tested forexpression of transgenes in the egg.

All documents cited in the above specification are herein incorporatedby reference. Various modifications and variations of the presentinvention will be apparent to those skilled in the art without departingfrom the scope and spirit of the invention. Although the invention hasbeen described in connection with specific preferred embodiments, itshould be understood that the invention as claimed should not be undulylimited to such specific embodiments. Indeed, various modifications ofthe described modes for carrying out the invention which are obvious tothose skilled in the art are intended to be within the scope of thefollowing claims.

1. A method comprising introducing a transgene encoding an exogenousprotein and a signal peptide into a chicken embryo; allowing the embryoto develop into a sexually mature male chicken; breeding the malechicken to a recipient female chicken; obtaining offspring from therecipient female which express the protein in tubular gland cells of theoviduct from which the protein is secreted; and recovering from an eggof an offspring a protein wherein the protein is exogenous to the eggand is encoded by the transgene.
 2. The method of claim 1 wherein thetransgene is contained between a 5′ LTR and 3′ LTR of a retroviralvector.
 3. The method of claim 1 wherein the exogenous protein isselected from the group consisting of antitrypsin, antithrombin III,collagen, factors VIII, IX, X, fibrinogen, insulin, lactoferrin, proteinC, tissue-type plasminogen activator, somatotrophin, cytokine, antibody,human growth hormone, immunotoxin and chymotrypsin.
 4. The method ofclaim 1 wherein the exogenous protein is present in egg white of theegg.
 5. The method of claim 1 wherein the transgene comprises aretroviral vector.
 6. The method of claim 5 wherein the retroviralvector is an ALV,
 7. A method comprising introducing a transgeneencoding a pharmaceutical protein and a signal peptide into a chickenembryo; allowing the embryo to develop into a sexually mature malechicken; breeding the male chicken to a recipient female chicken;obtaining offspring from the recipient female which express thepharmaceutical protein in tubular gland cells of the oviduct from whichthe protein is secreted; and recovering a pharmaceutical protein from anegg of an offspring wherein the pharmaceutical protein is exogenous tothe egg and is encoded by the transgene.
 8. The method of claim 7wherein the pharmaceutical protein is present in egg white of the egg.9. A method comprising introducing a transgene encoding an exogenouscytokine and a signal peptide into a chicken embryo; allowing the embryoto develop into a sexually mature male chicken; breeding the malechicken to a recipient female chicken; obtaining offspring from therecipient female which express the protein in tubular gland cells of theoviduct from which the protein is secreted; and recovering from an eggof an offspring a cytokine.
 10. The method of claim 9 wherein thecytokine is GM-CSF.
 11. The method of claim 9 wherein the cytokine isG-CSF.
 12. The method of claim 9 wherein the cytokine is erythropoietin.13. The method of claim 9 wherein the cytokine is interferon.
 14. Themethod of claim 9 comprising isolating the cytokine.